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1.
ChemMedChem ; 11(16): 1856-64, 2016 08 19.
Article in English | MEDLINE | ID: mdl-27061352

ABSTRACT

Previous modifications of the peptide sequence of human urotensin-II (U-II) led to the identification of two well-known ligands: P5U and urantide. These derivatives are considered to be the most representative agonist and antagonist, respectively, at the human urotensin receptor (UT). Optimization of P5U and urantide was carried out to stabilize specific conformations that may suggest new elements for discriminating agonist versus antagonist activity. We studied novel derivatives containing uncoded amino acids. In particular, the Tyr(9) residue of both P5U and urantide was replaced with nonaromatic hydrophobic bulky residues, as well as conformationally constrained aromatic moieties to generate eight novel derivatives. These analogues further contributed to determining the influence of such residues on binding affinity for and biological activity at UT. One of these eight peptides was also investigated by NMR spectroscopy and docking studies owing to its peculiar conformational properties and mode of interaction with UT. This structure-activity study is aimed at a more thorough examination of the role of tyrosine in modulating the agonism/antagonism of human U-II.


Subject(s)
Amino Acids/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Peptides/pharmacology , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Urotensins/chemistry , Urotensins/pharmacology , Amino Acids/chemistry , Animals , Dose-Response Relationship, Drug , Humans , Male , Molecular Structure , Peptides/chemistry , Rats , Rats, Wistar , Structure-Activity Relationship
2.
Bioorg Med Chem Lett ; 25(7): 1603-6, 2015 Apr 01.
Article in English | MEDLINE | ID: mdl-25746815

ABSTRACT

In the search for a new class of histone deacetylase inhibitors, we prepared a series of very simple benzofused hydroxamic acids to find an anchoring fragment of minimal molecular weight: they showed very good ligand efficiencies. Following these findings, classical fragment growing work was performed to increase binding energy and selective cytotoxicity. In the second phase of the work, information from the SARs of the benzothiophene series and data available in literature, we explored the in vitro pharmacological properties of the 6-substituted-7-fluoro-benzothiophene hydroxamates and the 5-susbtituted-benzofuran hydroxamates.


Subject(s)
Benzofurans/pharmacology , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Hydroxamic Acids/pharmacology , Thiophenes/pharmacology , Benzofurans/chemical synthesis , Benzofurans/chemistry , Dose-Response Relationship, Drug , HCT116 Cells , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/chemistry , Humans , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/chemistry , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/chemistry
3.
J Med Chem ; 57(14): 5965-74, 2014 Jul 24.
Article in English | MEDLINE | ID: mdl-24992374

ABSTRACT

We have optimized 1 (P5U) and urantide, two important ligands at the h-UT receptor, designing several analogues by the exchange of the Tyr9 residue with different unnatural aromatic amino acids. This study allowed us to discover novel ligands with improved activity. In particular, the replacement of the Tyr9 residue by (pCN)Phe or (pNO2)Phe within the urantide sequence led to compounds 13 (UPG-83) and 15 (UPG-95), respectively, which showed pure antagonist activity toward UT receptor in a rat aorta bioassay. More interestingly, the replacement of the Tyr9 in 1 sequence with the Btz or the (3,4-Cl)Phe residues led to superagonists 6 (UPG-100) and 10 (UPG-92) with pEC50 values at least 1.4 log higher than that of 1, being the most potent UT agonists discovered to date. Compounds 10 and 13 showed also a good stability in a serum proteolytic assay. These ligands represent new useful tools to further characterize the urotensinergic system in human physiopathology.


Subject(s)
Drug Discovery , Peptide Fragments/pharmacology , Peptides, Cyclic/pharmacology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/metabolism , Urotensins/pharmacology , Dose-Response Relationship, Drug , Humans , Ligands , Models, Molecular , Molecular Conformation , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Structure-Activity Relationship , Urotensins/chemical synthesis , Urotensins/chemistry
4.
Bioorg Med Chem Lett ; 23(14): 4091-5, 2013 Jul 15.
Article in English | MEDLINE | ID: mdl-23768910

ABSTRACT

In the search for a new class of histone deacetylase inhibitors, we prepared a series of simple benzofused hydroxamic acids to find an anchoring fragment of minimal molecular weight. These initial hits, all belonging to the benzothiophene class, showed very good ligand efficiencies. Following these findings, a classical fragment growing approach was performed to increase binding affinity and cytotoxicity.


Subject(s)
Histone Deacetylase Inhibitors/chemistry , Histone Deacetylases/chemistry , Hydroxamic Acids/chemistry , Thiophenes/chemistry , Cell Survival/drug effects , HCT116 Cells , Histone Deacetylase Inhibitors/metabolism , Histone Deacetylase Inhibitors/toxicity , Histone Deacetylases/metabolism , Humans , Hydroxamic Acids/metabolism , Hydroxamic Acids/toxicity , Protein Binding
5.
Bioorg Med Chem Lett ; 21(22): 6767-9, 2011 Nov 15.
Article in English | MEDLINE | ID: mdl-21978679

ABSTRACT

A series of N-substituted 4-alkylpiperidine hydroxamic acids, corresponding to the basic structure of histone deacetylase (HDAC) inhibitors (zinc binding moiety-linker-capping group) has been previously reported by our group. Linker length and aromatic capping group connection were systematically varied to find the optimal geometric parameters. A new series of submicromolar inhibitors was thus identified, which showed antiproliferative activity on HCT-116 colon carcinoma cells. We report here the second part of the strategy used in our research group to find a new class of HDAC inhibitors, namely the SAR study for the compounds bearing a sulfonyl group on the piperidine nitrogen. In the present work, we have considered both sulfonamides and sulfonyl ureas.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Piperidines/chemistry , Piperidines/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Colonic Neoplasms/drug therapy , Humans , Sulfones/chemistry , Sulfones/pharmacology
6.
Bioorg Med Chem Lett ; 21(8): 2305-8, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-21420859

ABSTRACT

We report here the strategy used in our research group to find a new class of histone deacetylase (HDAC) inhibitors. A series of N-substituted 4-alkylpiperazine and 4-alkylpiperidine hydroxamic acids, corresponding to the basic structure of HDAC inhibitors (zinc binding moiety-linker-capping group) has been designed, prepared, and tested for HDAC inhibition. Linker length and aromatic capping group connection were systematically varied to find the optimal geometric parameters. A new series of submicromolar inhibitors was thus identified, which showed antiproliferative activity on HCT-116 colon carcinoma cells.


Subject(s)
Antineoplastic Agents/chemistry , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylases/chemistry , Hydroxamic Acids/chemistry , Piperazines/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Humans , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacology , Piperazine , Structure-Activity Relationship
7.
J Med Chem ; 53(10): 4148-65, 2010 May 27.
Article in English | MEDLINE | ID: mdl-20408549

ABSTRACT

As part of a project aimed at the identification of a series of small, orally available antagonists for the hNK(2) receptor, starting from one of our capped dipeptide libraries, we succeeded in the chemical optimization of the first identified leads, finally producing a class of molecules with significant activity in our animal model after iv administration. We herein report the results of further chemical modifications made to reduce the overall peptide character of this series and the consequent improvement of their in vivo antagonist activity. The present work identified 6-methylbenzo[b]thiophene-2-carboxylic acid (1-[(S)-1-benzyl-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl]cyclopentyl)amide (10i), endowed with subnanomolar potency in all the in vitro tests and being highly potent and of long duration upon in vivo testing after both iv and id dosing.


Subject(s)
Amides/chemical synthesis , Furans/chemical synthesis , Piperazines/chemical synthesis , Receptors, Neurokinin-2/antagonists & inhibitors , Thiophenes/chemical synthesis , Amides/chemistry , Amides/pharmacology , Animals , Colon/drug effects , Colon/physiology , Furans/chemistry , Furans/pharmacology , Guinea Pigs , Humans , In Vitro Techniques , Intestinal Absorption , Male , Molecular Conformation , Muscle Contraction , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Piperazines/chemistry , Piperazines/pharmacology , Radioligand Assay , Receptors, Neurokinin-2/agonists , Stereoisomerism , Structure-Activity Relationship , Thiophenes/chemistry , Thiophenes/pharmacology , Urinary Bladder/drug effects , Urinary Bladder/physiology
8.
Expert Opin Investig Drugs ; 18(12): 1843-64, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19938899

ABSTRACT

Tachykinins (TKs) are small peptides widely distributed in the central and peripheral nervous systems where they act as neurotransmitters. Potent and selective TKs antagonists have been developed in the last 20 years and many efforts have been made to prove their efficacy in the treatment of various diseases. Herein the most prominent results in the clinical development are reported and discussed. For aprepitant, the only compound of this class to have been launched to date, results of clinical studies and postmarketing cost-effectiveness data for the treatment of chemotherapy-induced emesis are discussed. The field is still well active, as currently proof-of-concept studies for indications initially missed (i.e., depression) are ongoing and new targets are under investigation.


Subject(s)
Antiemetics/therapeutic use , Neurokinin-1 Receptor Antagonists , Receptors, Neurokinin-2/antagonists & inhibitors , Receptors, Neurokinin-3/antagonists & inhibitors , Aprepitant , Asthma/drug therapy , Clinical Trials as Topic , Gastrointestinal Diseases/drug therapy , Humans , Mental Disorders/drug therapy , Morpholines/therapeutic use , Piperazines/therapeutic use , Piperidines/therapeutic use , Sleep Initiation and Maintenance Disorders/drug therapy
9.
J Med Chem ; 52(13): 3927-40, 2009 Jul 09.
Article in English | MEDLINE | ID: mdl-19432421

ABSTRACT

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of hU-II termed P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized several analogues of P5U and urantide in which the Asp(4) residue in N-terminus position was replaced with coded and noncoded amino acids. The replacement of the Asp(4) residue by Tic led to an analogue, compound 14, more potent as antagonist (pK(B) = 8.94) compared to urantide. Furthermore, a different SAR was observed for the P5U compared to the urantide analogues. NMR and docking studies revealed a different binding mode for the agonist and antagonist ligands which could explain the observed SAR.


Subject(s)
Oligopeptides/chemistry , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Cell Line , Computer Simulation , Humans , Ligands , Magnetic Resonance Spectroscopy , Oligopeptides/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Protein Binding , Receptors, G-Protein-Coupled/chemistry , Structure-Activity Relationship , Urotensins/chemistry , Urotensins/pharmacology
10.
J Med Chem ; 50(3): 550-65, 2007 Feb 08.
Article in English | MEDLINE | ID: mdl-17266207

ABSTRACT

Recently we reported on the design and synthesis of a novel class of selective nonpeptide bradykinin (BK) B2 receptor antagonists (J. Med. Chem. 2006, 3602-3613). This work led to the discovery of MEN 15442, an antagonist with subnanomolar affinity for the human B2 receptor (hB2R), which also displayed significant and prolonged activity in vivo (for up to 210 min) against BK-induced bronchoconstriction in the guinea-pig at a dose of 300 nmol/kg (it), while demonstrating only a slight effect on BK-induced hypotension. Here we describe the further optimization of this series of compounds aimed at maximizing the effect on bronchoconstriction and minimizing the effect on hypotension, with a view to developing topically delivered drugs for airway diseases. The work led to the discovery of MEN 16132, a compound which, after intratracheal or aerosol administration, inhibited, in a dose-dependent manner, BK-induced bronchoconstricton in the airways, while showing minimal systemic activity. This compound was selected as a preclinical candidate for the topical treatment of airway diseases involving kinin B2 receptor stimulation.


Subject(s)
Bradykinin B2 Receptor Antagonists , Bronchodilator Agents/chemical synthesis , Ornithine/analogs & derivatives , Sulfonamides/chemical synthesis , Animals , Blood Pressure/drug effects , Bronchoconstriction/drug effects , Bronchodilator Agents/chemistry , Bronchodilator Agents/pharmacology , CHO Cells , Cricetinae , Cricetulus , Drug Design , Guinea Pigs , Humans , Ileum/drug effects , Ileum/physiology , In Vitro Techniques , Inositol Phosphates/biosynthesis , Male , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Ornithine/chemical synthesis , Ornithine/chemistry , Ornithine/pharmacology , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacology
11.
J Med Chem ; 49(12): 3602-13, 2006 Jun 15.
Article in English | MEDLINE | ID: mdl-16759102

ABSTRACT

We recently published the extensive in vivo pharmacological characterization of MEN 16132 (J. Pharmacol. Exp. Ther. 2005, 616-623; Eur. J. Pharmacol. 2005, 528, 7), a member of the sulfonamide-containing human B(2) receptor (hB(2)R) antagonists. Here we report, in detail, how this family of compounds was designed, synthesized, and optimized to provide a group of products with subnanomolar affinity for the hB(2)R and high in vivo potency after topical administration to the respiratory tract. The series was designed on the basis of indications from the X-ray structures of the key structural motifs A and B present in known antagonists and is characterized by the presence of an alpha,alpha-dialkyl amino acid. The first lead (17) of the series was submitted to extensive chemical work to elucidate the structural requirements to increase hB(2) receptor affinity and antagonist potency in bioassays expressing the human B(2) receptor (hB(2)R). The following structural features were selected: a 2,4-dimethylquinoline moiety and a piperazine linker acylated with a basic amino acid. The representative lead compound 68 inhibited the specific binding of [(3)H]BK to hB(2)R with a pKi of 9.4 and antagonized the BK-induced inositolphosphate (IP) accumulation in recombinant cell systems expressing the hB(2)R with a pA(2) of 9.1. Moreover, compound 68 when administered (300 nmol/kg) intratracheally in the anesthetized guinea pig, was able to significantly inhibit BK-induced bronchoconstriction for up to 120 min after its administration, while having a lower and shorter lasting effect on hypotension.


Subject(s)
Bradykinin B2 Receptor Antagonists , Quinolines/chemical synthesis , Sarcosine/analogs & derivatives , Sulfonamides/chemical synthesis , Animals , Bradykinin/metabolism , Bronchoconstrictor Agents/chemical synthesis , Bronchoconstrictor Agents/chemistry , Bronchoconstrictor Agents/pharmacology , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Drug Design , Guinea Pigs , Humans , Hypotension/chemically induced , Inositol Phosphates/biosynthesis , Models, Molecular , Molecular Structure , Piperazine , Piperazines/chemistry , Quinolines/chemistry , Quinolines/pharmacology , Radioligand Assay , Receptor, Bradykinin B2/metabolism , Sarcosine/chemical synthesis , Sarcosine/chemistry , Sarcosine/pharmacology , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacology
12.
Basic Clin Pharmacol Toxicol ; 98(5): 488-95, 2006 May.
Article in English | MEDLINE | ID: mdl-16635108

ABSTRACT

Adenosine 5'-triphosphate (ATP) may be an important neurotransmitter in the gastrointestinal tract. The present study examined the motor effects of exogenous ATP on longitudinally-oriented preparations of the guinea-pig isolated ileum and the influence of drugs on the ATP-induced responses. High micromolar concentrations of ATP caused two types of contraction, a phasic, cholinergic response and a tonic, tetrodotoxin-resistant contraction. The phasic contraction was reduced by hexamethonium (5x10(-5) M), but left uninfluenced by capsaicin tachyphylaxis or tachyphylaxis to alpha,beta-methylene ATP. The tonic response was resistant to atropine, hexamethonium, capsaicin, omega-conotoxin GVIA, or pretreatment with alpha,beta-methylene ATP. Both types of ATP-induced contraction were diminished or abolished by the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 3x10(-6) and 3x10(-5) M, respectively). In the precontracted, atropine-treated ileum ATP (10(-6)-10(-4) M) caused guanethidine-resistant relaxation. This response was not influenced by tetrodotoxin, omega-conotoxin GVIA, or NG-nitro-L-arginine, but was abolished by apamin (10(-7) M), and inhibited by PPADS (3x10(-5) M) or reactive blue 2 (10(-5) M), in a surmountable manner. A high degree of tachyphylaxis was observed with the relaxant effect of ATP (10(-5)-10(-4) M). A high concentration (3x10(-4) M) of PPADS failed to influence ileum contractions to exogenous acetylcholine or histamine. It is concluded that, in addition to its direct contractile action in the guinea-pig ileum, ATP can activate (partly preganglionic) cholinergic neurones, an effect whose mechanism is largely different from that of alpha,beta-methylene ATP. ATP also causes relaxation by a direct, probably P2Y-receptor-mediated effect on the smooth muscle. All motor effects of ATP are inhibited by the antagonist PPADS.


Subject(s)
Adenosine Triphosphate/pharmacology , Ileum/drug effects , Motor Neurons/physiology , Pyridoxal Phosphate/analogs & derivatives , Receptors, Purinergic P2/drug effects , Acetylcholine/pharmacology , Animals , Atropine/pharmacology , Dose-Response Relationship, Drug , Guinea Pigs , Histamine/pharmacology , Ileum/innervation , Ileum/metabolism , In Vitro Techniques , Muscarinic Antagonists/pharmacology , Muscle Contraction , Muscle Relaxation , Muscle, Smooth/drug effects , Muscle, Smooth/innervation , Muscle, Smooth/metabolism , Pyridoxal Phosphate/pharmacology , Receptors, Purinergic P2/metabolism
13.
J Med Chem ; 48(23): 7290-7, 2005 Nov 17.
Article in English | MEDLINE | ID: mdl-16279788

ABSTRACT

Urotensin II (U-II) is a disulfide bridged peptide hormone recently identified as the ligand of a G-protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-cyclo[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of hU-II termed P5U and the compound termed urantide, which is the most potent UT receptor peptide antagonist described to date. Our previous conformational studies showed that hU-II and its analogues with agonist activity adopt a well-defined type II' beta-hairpin structure in anisotropic SDS membrane-like environment. This structural arrangement allows tight contact among the Trp7, Lys8, and Tyr9 side chains, which is fundamental to obtain full agonist activity. Here, we report an extensive SAR study on new analogues with agonist/antagonist activity on UT receptor. We investigated their biological activity and performed a conformational analysis by spectroscopic and computational methods. Our goal is to obtain a structure-based model able to explain the agonist/antagonist functional switching of these ligands.


Subject(s)
Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Urotensins/chemical synthesis , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiology , CHO Cells , Chromatography, High Pressure Liquid , Cricetinae , Cricetulus , Humans , In Vitro Techniques , Magnetic Resonance Spectroscopy , Male , Micelles , Models, Molecular , Muscle Relaxation/drug effects , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Protein Structure, Secondary , Radioligand Assay , Rats , Rats, Wistar , Sodium Dodecyl Sulfate , Structure-Activity Relationship , Urotensins/chemistry , Urotensins/pharmacology
14.
Life Sci ; 76(24): 2773-82, 2005 Apr 29.
Article in English | MEDLINE | ID: mdl-15808879

ABSTRACT

Moderate concentrations of the sensory stimulant drug capsaicin caused relaxation in human and animal intestinal circular muscle preparations (guinea-pig proximal, mouse distal colon, human small intestine and appendix) in vitro. With the exception of the guinea-pig colon, the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 10(-4) M) strongly inhibited the relaxant effect of capsaicin. Tetrodotoxin, an inhibitor of voltage-sensitive Na+ channels failed to significantly reduce the inhibitory effect of capsaicin in the guinea-pig colon, human ileum and appendix; it caused an approximately 50% reduction in the mouse colon. The relaxant effect of capsaicin was strongly reduced in colonic preparations from transient receptor potential vanilloid type (TRPV1) receptor knockout mice as compared to their wildtype controls. It is concluded that nitric oxide, possibly of sensory origin, is involved in the relaxant action of capsaicin in the circular muscle of the mouse and human intestine.


Subject(s)
Capsaicin/antagonists & inhibitors , Colon/metabolism , Muscle Relaxation/drug effects , Muscle, Smooth/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitroarginine/pharmacology , Tetrodotoxin/pharmacology , Animals , Capsaicin/pharmacology , Guinea Pigs , Humans , Mice , Mice, Inbred Strains , Muscle, Smooth/drug effects
15.
Br J Pharmacol ; 139(3): 641-51, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12788824

ABSTRACT

(1) The anticancer anthracycline doxorubicin (DOX) causes cardiomyopathy upon chronic administration. There is controversy about whether DOX acts directly or after conversion to its secondary alcohol metabolite DOXol. Here, the role of secondary alcohol metabolites was evaluated by treating rats with cumulative doses of DOX or analogues--like epirubicin (EPI) and the novel disaccharide anthracycline MEN 10755--which were previously shown to form less alcohol metabolites than DOX when assessed in vitro. (2) DOX induced electrocardiographic and haemodynamic alterations, like elongation of QalphaT or SalphaT intervals and suppression of isoprenaline-induced dP/dt increases, which developed in a time-dependent manner and were accompanied by cardiomegaly, histologic lesions and mortality. EPI caused less progressive or severe effects, whereas MEN 10755 caused essentially no effect. (3) DOX and EPI exhibited comparable levels of cardiac uptake, but EPI formed approximately 60% lower amounts of its alcohol metabolite EPIol at 4 and 13 weeks after treatment suspension (P<0.001 vs DOX). MEN 10755 exhibited the lowest levels of cardiac uptake; hence, it converted to its alcohol metabolite MEN 10755ol approximately 40% less efficiently than did EPI to EPIol at either 4 or 13 weeks. Cardiotoxicity did not correlate with myocardial levels of DOX or EPI or MEN 10755, but correlated with those of DOXol or EPIol or MEN 10755ol (P=0.008, 0.029 and 0.017, respectively). (4) DOX and EPI inactivated cytoplasmic aconitase, an enzyme containing an Fe-S cluster liable to disassembly induced by anthracycline secondary alcohol metabolites. DOX caused greater inactivation of aconitase than EPI, a finding consistent with the higher formation of DOXol vs EPIol. MEN 10755 did not inactivate aconitase, which was because of both reduced formation and impaired reactivity of MEN 10755ol toward the Fe-S cluster. Aconitase inactivation correlated (P<0.01) with the different levels of cardiotoxicity induced by DOX or EPI or MEN 10755. (5) These results show that (i) secondary alcohol metabolites are important determinants of anthracycline-induced cardiotoxicity, and (ii) MEN 10755 is less cardiotoxic than DOX or EPI, a behaviour attributable to impaired formation and reactivity of its alcohol metabolite.


Subject(s)
Anthracyclines/metabolism , Anthracyclines/toxicity , Antineoplastic Agents/metabolism , Antineoplastic Agents/toxicity , Myocardium/metabolism , Animals , Anthracyclines/administration & dosage , Antineoplastic Agents/administration & dosage , Body Weight/drug effects , Body Weight/physiology , Heart Atria/drug effects , Heart Atria/metabolism , Heart Rate/drug effects , Heart Rate/physiology , Heart Ventricles/drug effects , Heart Ventricles/metabolism , Heart Ventricles/pathology , Male , Myocardium/pathology , Organ Size/drug effects , Organ Size/physiology , Rats , Rats, Sprague-Dawley
16.
Mol Biotechnol ; 23(3): 189-202, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12665690

ABSTRACT

We describe an expression system for high-yield production of recombinant soluble human FasL (rsh- FasL) in CHO cells. After one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 microg/L x 10(6) cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures. The protein secreted into growth medium was purified by immunoaffinity. The rsh-FasL thus obtained was further fractionated by gel filtration and a form of approx 140 kDa was isolated and characterized. Mass spectral analysis yielded a main peak of 28,321.15 Da, while, although to a lesser extent, dimeric and trimeric forms were also detected according to the described oligomerized state of native FasL. Our procedure permits consistent production of biologically active rsh-FasL as shown in tests on FasL-sensitive cells and in in vitro binding assays.


Subject(s)
CHO Cells/metabolism , Gene Expression Regulation , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Amino Acid Sequence , Animals , CHO Cells/physiology , Cells, Cultured , Cricetinae , Fas Ligand Protein , Gene Amplification , Humans , Ligands , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/isolation & purification , Molecular Sequence Data , Mutagenesis, Site-Directed , Quality Control , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Transfection
17.
J Pharmacol Exp Ther ; 304(1): 272-6, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12490601

ABSTRACT

Tachykinins have been implicated in inflammatory responses such as those occurring in inflammatory bowel disease. Accordingly, we investigated the effect of a selective neurokinin (NK) 2 receptor antagonist, nepadutant, on proto-oncogene expression in the L(6)-S(1) spinal cord as well as in dorsal root ganglion (DRG) neurons after either non-noxious colorectal distension (CRD) or trinitrobenzenesulfonic acid (TNBS)-induced colitis in the adult rat. In both preparations, c-fos was expressed in similar spinal cord regions, including medial and lateral dorsal horn, dorsal commissure (DCM; laminae X above the central canal), and the sacral parasympathetic nucleus (SPN, laminae V-VII). However, TNBS-induced colitis produced significantly larger numbers (8-10-fold increase over control) of Fos-positive spinal cord neurons. In addition, there was also a significant increase (3-4-fold) in the number of Jun-positive colon DRG neurons after colitis compared with CRD. Nepadutant had no significant effect on proto-oncogene expression induced by CRD in either spinal cord neurons or DRG neurons. In contrast, nepadutant significantly decreased (70%) the number of Fos-positive neurons in dorsal horn, DCM, and SPN spinal cord regions and significantly decreased (75%) the number of Jun-positive DRG neurons after TNBS-induced irritation of the colon. These findings indicate that nepadutant suppresses the responses of colonic afferent neurons to nociceptive stimuli and that NK2 receptor antagonists may be beneficial in the treatment of sensory symptoms of colitis.


Subject(s)
Colitis/metabolism , Gene Expression/drug effects , Genes, Immediate-Early/genetics , Peptides, Cyclic/pharmacology , Receptors, Neurokinin-2/antagonists & inhibitors , Trinitrobenzenesulfonic Acid , Animals , Capsaicin/pharmacology , Colitis/chemically induced , Female , Ganglia, Spinal/metabolism , Genes, fos/drug effects , Genes, jun/drug effects , Irritants , Neurons/drug effects , Neurons/metabolism , Physical Stimulation , Rats , Rats, Sprague-Dawley , Spinal Cord/metabolism
18.
Bioorg Med Chem ; 10(12): 3731-9, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12413830

ABSTRACT

Human urotensin II (hU-II; H-Glu-Thr-Pro-Asp-cyclo[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) is a disulfide bridged undecapeptide recently identified as the ligand of an orphan G protein-coupled receptor. hU-II has been described as the most potent vasoconstrictor compound identified to date. With the aim of replacing the disulfide bridge by a chemically more stable moiety, we have synthesized and tested a series of lactam analogues of hU-II minimum active fragment, that is hU-II(4-11). The contractile activity of the synthetic analogues on the rat isolated thoracic aorta was found to be dependent upon the dimension of the lactam bridge. The most active peptide, H-Asp-cyclo[Orn-Phe-Trp-Lys-Tyr-Asp]-Val-OH (3), is approximately 2 logs less potent than hU-II (pD(2)=6.3 vs 8.4). A conformational analysis in solution of the active peptide 3, one of the inactive analogues, and hU-II was performed, using NMR and molecular modelling techniques. A superposition of the calculated structures of hU-II and 3 clearly shows that three out of four key residues (i.e., Phe(6), Lys(8) and Tyr(9)) maintain the same side- chain orientation, while the fourth one, Trp(7), cannot be superimposed. This observation could explain the reduced biological activity of the synthetic analogue.


Subject(s)
Lactams/pharmacology , Urotensins/chemistry , Vasoconstrictor Agents/chemical synthesis , Animals , Aorta/drug effects , Drug Design , Drug Stability , Humans , Lactams/chemical synthesis , Lactams/chemistry , Male , Models, Molecular , Molecular Conformation , Nuclear Magnetic Resonance, Biomolecular , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Rats , Rats, Wistar , Structure-Activity Relationship , Urotensins/pharmacology , Vasoconstrictor Agents/chemistry , Vasoconstrictor Agents/pharmacology
19.
Int J Cancer ; 102(5): 476-82, 2002 Dec 10.
Article in English | MEDLINE | ID: mdl-12432549

ABSTRACT

The new disaccharide anthracycline MEN 10755 induces activation of both NF-kappaB and p53 transcription factors in A2780 cells. Nevertheless, pharmacologic inhibition of NF-kappaB activation does not modify the sensitivity of A2780 cells to MEN 10755 treatment. To better characterize the role of NF-kappaB in MEN 10755-induced cytotoxicity, we analyzed the expression of a number of genes that are known to be regulated by NF-kappaB. None of these genes is modified by MEN 10755 treatment. On the contrary, our results suggest that the p53 DNA damage-responsive pathway is fully activated in A2780 cells, several genes controlled by p53 being up- or downregulated according to the described action of p53 on their promoters. Thus, in the A2780 cell line, the role of p53 in transducing the DNA-damage signal appears to be relevant, whereas NF-kappaB, although activated, appears to be nonfunctional. Other human carcinoma cell lines besides A2780 activate NF-kappaB DNA binding in response to MEN 10755 treatment, but again, this binding does not always lead to target gene activation. These results suggest that other factors, tumor type-specific and different from mere activation, could influence NF-kappaB transcriptional activity. Therefore, care should be taken when considering the pharmacologic inhibition of NF-kappaB as a means to improve anticancer therapy efficacy.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma/metabolism , Disaccharides/pharmacology , Doxorubicin/analogs & derivatives , Doxorubicin/pharmacology , NF-kappa B/metabolism , Gene Expression Regulation, Neoplastic , Humans , NF-kappa B/antagonists & inhibitors , Transcriptional Activation , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism
20.
J Med Chem ; 45(20): 4391-4, 2002 Sep 26.
Article in English | MEDLINE | ID: mdl-12238917

ABSTRACT

Replacing Cys(5) by Pen (penicillamine, beta,beta-dimethylcysteine) in the cyclic C-terminal U-II octapeptide, U-II(4-11), we have obtained a potent urotensin II (U-II) receptor agonist. Conformational analysis of solution NMR data indicated that the putative biologically active conformation of U-II is stabilized by introduction of a Pen residue. To the best of our knowledge, this is the most potent U-II receptor agonist reported to date.


Subject(s)
Disulfides/chemistry , Oligopeptides/chemical synthesis , Penicillamine/chemistry , Peptides, Cyclic/chemical synthesis , Receptors, Cell Surface/agonists , Receptors, G-Protein-Coupled , Animals , Binding, Competitive , CHO Cells , Cricetinae , Cysteine/chemistry , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Oligopeptides/chemistry , Oligopeptides/pharmacology , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Radioligand Assay , Structure-Activity Relationship
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